[RASMB] MALLS

[John Philo]

Joel and RASMB,

1. This is definitely a useful technology for proteins, and indeed I believe
the majority of the instruments are now sold for protein work rather than
polymers. This is an easy and quick way to determine whether the native
state of a protein is a monomer, dimer,... and to determine the
stoichiometry of protein-protein complexes. 

You can find some examples of how we apply this technology on the A.P.L.
website www.ap-lab.com and in our 1996 review article (which can be
downloaded from there). 

You also might want to see the Biophysics Resource Facility site at Yale,
where they have some background info and test data.
http://info.med.yale.edu/wmkeck/biophysics/Lsmemoa.htm#HHMI%20site%20index%2
0(LSmemoa)

2. In my experience the accuracy is 2-3% from ~2-1000 kDa. For lower mass
species the sensitivity is lower so you need to inject more protein to get
good signal/noise.

3. I only have direct experience with the Wyatt miniDAWN. Those have been
extremely reliable and their technical support and training is very good. 

4. It is definitely best to have an RI detector in series with the LS
detector. This makes the calibration nearly the same for all polypeptides.
Without that your mass accuracy is only as good as the absolute accuracy of
your extinction coefficient, and you may well not know the extinction
coefficients of all the peaks coming off your column. 

John Philo
Alliance Protein Laboratories


-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of Joel Mackay
Sent: Monday, February 17, 2003 12:44 AM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] MALLS


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Hi all,
We are currently considering the purchase of a multiangle laser light 
scattering instrument for getting a snapshot of protein molecular weight - 
we intend to hook up to one of our chromatography systems. [Of course it 
isn't intended as a replacement for our venerable XLA... - we just thought 
it would be handy for getting a feel for molecular weight during the 
chromatography stage].

I must confess i haven't really got any experience with this technology, 
but i thought at least some of the people in this forum might, and i hoped 
i could get answers to a couple of questions...

1. What is the general feeling about this method for looking at proteins - 
i know quite a few people use it in chemical polymer applications, but 
their problems are often a bit different (branching etc), and their 
polymers are often much bigger. Is it useful? We are not so interested in 
shape (so are not bothering with dynamic light scattering) - more in 
molecular mass.

2. What sort of masses can be measured with confidence on an instrument 
like the mini-Dawn from Wyatt - we often deal with proteins that are ~5-10
kDa.

3. Wyatt seems to be the major player in the field, but i have been getting 
info from Precision Detectors as well. Does anyone have any comments 
(printable!) to make about either manufacturer's instruments etc?

4. Do people tend to use a refractometer to measure refractive indices, or 
use calculated values?

Any comments most appreciated!

cheers
joel


*****************************************************************
Dr Joel Mackay
Senior Lecturer
School of Molecular and Microbial Biosciences
Building G08
University of Sydney,
Sydney, NSW 2006
Australia
ph +61-2-9351-3906
fax +61-2-9351-4726
WWW: http://www.mmb.usyd.edu.au/mackay/
Sydney Protein Group Website:
http://www.mmb.usyd.edu.au/spg/
****************************************************************

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